This proposal is in response to RFA-A1-14-027: Development of Sample Sparing Assays for Monitoring Immune Responses (U24). We will develop ultrasensitive assays for 20 cytokines that have largely been unmeasurable in serum and plasma in the past. The assays will be carried out on a fully automated instrument in user-defined multiplexed combinations, at single-digit fg/mL sensitivity, using only 55 uL of sample per measurement of a set of ten analytes (instrument dead volume included). After analytically validating the system, we will demonstrate its utility in collaboration with academic groups in the NIAID DAIT network. We will implement sample sparing by two means: first by multiplexing (ten assays per well), which reduces sample use per assay by an order of magnitude; second, by employing a novel ultrasensitive assay technology that improves assay sensitivity approximately 100-fold. We have demonstrated detection limits of 1 fg/mL or better for several cytokine assays using this technology (Glezer et al., 2014). This increased sensitivity can be used to measure cytokines in samples where levels are too low to be detectable using current technologies. For many applications, the single-digit fg/mL sensitivity will allow the sample to be diluted, resulting in further reduction in the volume of sample required. The developed assays will be available on the large installed base of over 1,000 MSD instruments, with manual assay protocols. Additionally, the assays will be available on a fully automated analyzer, which not only provides the research community with a convenient, labor saving tool, but by also improves reproducibility and robustness of the assays. In the first two years of this project, we will develop 16 ultrasensitive cytokine assays with detection limits of 10 fg/mL or better (an additional four assays are already available). In year three, we will demonstrate multiplexing in a manual format. In year four, these 20 ultrasensitive assays will be transferred to an automated platform (developed under independent funding). In the last year, an analytical performance verification study and an external validation study will be performed.